Hypoglycemia remains the major factor limiting the use of intensified insulin therapy that has been shown to prevent complications in type 1 diabetes (T1DM). This MERIT extension proposal continues studies focused on the mechanisms that trigger glucose counterregulation and lead to the development of hypoglycemiaassociated autonomic failure (HAAF) and hypoglycemia unawareness. The protocols utilize unique rodent models to test specific hypotheses, and where possible include studies in patients with T1DM. We will evaluate the molecular mechanisms used by glucose-sensing neurons in the ventromedial hypothalamus (VMM) to activate counterregulatory responses. Specifically, we test the hypothesis that hypoglycemia activates a coordinated VMH signal that results from the opening of KATP channels in glucose-sensing neurons causing a drop in GABAergic inhibitory tone that promotes the activation of VMH glucose-inhibited neurons by the fuel sensing enzyme, AMP-kinase. This model of VMH glucose sensing will serve as a starting point for studies directed at altering VMH gene expression in rodents to evaluate putative mechanisms responsible for HAAF, including the role of increased GABA or CRF2 receptor inhibitory input or decreased AMP-kinase activity within the VMH. The potential therapeutic value of diazoxide will also be tested in intensively treated T1DM patients with suppressed glucose counterregulation. The influence of insulin per se on VMH glucose sensing will be determined. We will test the hypothesis that insulin acts within the VMH to tonically suppress glucagon secretion in the postaborptive state and to diminish the release of glucagon during hypoglycemia. In addition, we will continue study insulin's effect on hippocampus function, specifically whether its beneficial effect on memory function is altered by chronic hyperglycemia. We will also examine the mechanisms for improved cognitive performance in rats after long-term exposure to recurrent hypoglycemia using MR spectroscopy as well as genomic and proteomic analyses. Finally, we will evaluate how brain activation patterns assessed by fMRI and hypothalamic blood flow are affected by acute hypoglycemia in T1DM patients with severe hypoglycemia unawareness as compared to controls and if such changes are reduced by medium chain fatty acids. The long-term goal is to develop novel clinical strategies aimed at minimizing the risk of insulin-induced hypoglycemia in patients with T1DM.